Influences of elevational gradient on flower size and number of Gentiana lawrencei var. farreri

Abstract Plants can adapt to environmental changes by adjusting their functional traits and biomass allocation. The size and number of flowers are functional traits related to plant reproduction. Life history theory predicts that there is a trade‐off between flower size and number, and the trade‐off can potentially explain the adaptability of plants. Elevation gradients in mountains provide a unique opportunity to test how plants will respond to climate change. In this study, we tried to better explain the adaptability of the alpine plant Gentiana lawrencei var. farreri in response to climate change. We measured the flower size and number, individual size, and reproductive allocation of G. lawrencei var. farreri during the flowering period along an elevation gradient from 3200 to 4000 m, and explored their relationships using linear mixed‐effect models and the structural equation model. We found that with elevation increasing, individual size and flower number decreased and flower size increased, while reproductive allocation remained unchanged. Individual size positively affected flower number, but was not related to flower size; reproductive allocation positively affected flower size, but was not related to flower number; there is a clear trade‐off between flower size and number. We also found that elevation decreased flower number indirectly via directly reducing individual size. In sum, this study suggests that G. lawrencei var. farreri can adapt to alpine environments by the synergies or trade‐offs among individual size, reproductive allocation, flower size, and flower number. This study increases our understanding of the adaptation mechanisms of alpine plants to climate change in alpine environments.


| INTRODUC TI ON
Plants can adapt to environmental changes by adjusting their functional traits and reproductive allocation (Atkin et al., 2015;Körner, 2003).Floral display in terms of flower size and number has influences on pollinator visitation rate and seed production (Bell, 1985;Sandring & Ågren, 2009;Wu et al., 2023).Therefore, floral display is an important reproductive trait related to fitness (Sargent et al., 2007), and is often used to study plant evolution and adaptation mechanisms (Sandring & Ågren, 2009;Zhang et al., 2017).Plants have evolved diverse flower displays to adapt to environmental changes and extreme environments (such as highaltitude environments) (Körner, 2003;Kuppler & Kotowska, 2021;Zhang et al., 2017).
Life history theory suggests that a plant allocates its resources to different needs, which lead to trade-offs between different functions and activities due to finite resources of a plant (Silvertown & Charlesworth, 2009).That is to say, an increase in the amount of resources invested in a certain function (trait) will inevitably lead to a decrease in the amount of resources invested in other functions (traits).Reproductive allocation controls the balance between plant survival and reproduction, and is one of the core theories of plant life history (Friedman, 2020).Plants tend to choose optimal reproductive allocation to achieve maximum fitness (Wong & Ackerly, 2005).Nonbiological (e.g., elevation) and biological (e.g., competition) factors are generally believed to shift the balance between vegetative growth and reproduction (Friedman, 2020).In addition, within reproduction function, there should be a trade-off between flower size and number based on hierarchical resource allocation (Obeso, 2002;Richards et al., 2009).That is to say, plants can either produce fewer and larger flowers or produce more and smaller flowers.Life history theory also suggests that the evolution of floral display is constrained by the trade-off between flower size and number (Sargent et al., 2007).
Although its existence has a convincing theoretical basis, empirical evidence of the trade-off between flower size and number has always been elusive (reviewed by Caruso et al., 2012 andSargent et al., 2007).For instance, some empirical studies have found no relationship between flower size and number (Ashman & Majetic, 2006;Caruso et al., 2012;Worley & Barrett, 2001).
Elevation gradients in mountains are usually used as a substitution to test how plants will respond to climate change (Li et al., 2016).
High-altitude areas have harsh environments, such as low temperatures, short growing season, and variability and poor predictability of climate, which not only affect the growth and development of alpine plants but also affect the interaction between plants and animals (Körner, 2003).Due to unfavorable growth conditions, plant size generally decreases with increasing elevation (Coomes & Allen, 2007;Kiełtyk, 2021;Sigdel et al., 2023).It is generally believed that in highaltitude environments, plant reproduction and population persistence suffer greater pressure, resulting in an increase in plant reproductive allocation as elevation increases (Fabbro & Körner, 2004;Rathee et al., 2021).The individual size of plants can reflect the total amount of plant resources.The increase in both total plant resources and reproductive allocation can potentially increase the number and size of flowers (Zhang, 2004).One might predict then that elevation could indirectly influence flower size and number via individual size and reproductive allocation (Figure 1).
Numerous researches have shown that the diversity and abundance of pollinating insects and the number of flower-visiting insects per plant decreased with increasing elevation (Adedoja et al., 2020;Lefebvre et al., 2018).Compared to low-altitude areas, lower temperatures in high-altitude environments can weaken the activity of flowervisiting insects (Arroyo et al., 1985;Bingham & Ortner, 1998;Goodwin et al., 2021).Therefore, many outcrossing alpine plants may suffer from strong pollination limitation (Duan et al., 2007;Sun et al., 2018), which may reduce the success rate of outcrossing plant reproduction.
In order to adapt to the special environment of alpine regions and attract more insects to visit flowers, plants must make some efforts to potentially produce larger flowers (Kiełtyk, 2018;Körner, 2003).
Gentiana species are typical alpine plants, with the Qinghai-Tibet Plateau and its surrounding areas as their distribution and differentiation centers (Ho & Liu, 2001).As a common species of the Gentiana genus, Gentiana lawrencei var.farreri is a perennial herbaceous plant endemic to the Qinghai-Tibet Plateau, a dominant species in alpine meadows (Ho & Liu, 2001) and an important traditional medicinal plant (Yang et al., 1991).Elevation is the key determinant of bioclimatic gradients on the Qinghai-Tibet Plateau (Qi et al., 2015;Sigdel et al., 2023;Zhang et al., 2022).The unique alpine environmental conditions of the Qinghai-Tibet Plateau provide a relatively good platform for studying the adaptability and evolutionary mechanisms of plant resource allocation (Duan et al., 2007;Hou et al., 2024).
Here, we chose the typical alpine plant G. lawrencei var.farreri in the

| Study area
The experiment was carried out at the Qinghai Haibei National Field Research Station of Alpine Grassland Ecosystem in China (37°37′ N, 101°12′ E).The station is situated in the northeast of the Qinghai-Tibet Plateau.The region has a typical plateau continental climate, with long and cold winters and short and cool summers (Zhang et al., 2019).In September 2023, we sampled G. lawrencei var.farreri during flowering every 100-300 m along a 3200-4000 m elevational gradient (Table S1; see photographs of the habitat of G. lawrencei var. farreri in Figure S1) on the south slope of the Qilian Mountains in Qinghai, China.A long-term field study (Li et al., 2016) in the same site reported that annual average soil temperatures at 5 cm depth were 3.9, 2.5, 2.0, and 0.4 °C, and annual average soil moistures at 20 cm depth were 11.8, 11.3, 12.7, and 10.2% at 3200, 3400, 3600, and 3800 m, respectively.

| Study species
G. lawrencei var.farreri is a perennial herbaceous plant with a height of 5-12 cm.The flower branches are numerous, clustered, and scattered.The rosette leaves are extremely underdeveloped and lanceolate, with a length of 4-6 mm and a width of 2-3 mm.One flower grows at the top of the branch, and is inverted cone-shaped, with a sky-blue upper part and a yellow green lower part, and with blue stripes and a length of 4.5-6.0cm.It is mainly distributed in the northeastern Qinghai-Tibetan Plateau from 2400 to 4000 m above sea level (Ho & Liu, 2001).Its peak flowering period is in mid-September, and its main pollinating insects are Bombus kashmirensis and B. sushikini (Hou et al., 2009).

| Sampling and data measurement
We selected the G. lawrencei var.farreri population that met the sampling requirements (including at least 30 healthy, fully developed flowering individuals) every 100-300 m along the elevational gradient.We then randomly selected 12 apparently healthy, fully developed flowering individuals from the population of G. lawrencei var.farreri at each elevation (3200, 3500, 3750, 3900, and

| Data analyses
We applied standard linear models to examine the relationships be- The standard linear model, linear mixed-effect model and SEM were conducted using the packages base (Tollefson, 2019), lmerTesT (Bates et al., 2015), and piecewiseSEM (Lefcheck, 2016)   affected flower size (p < .001; Figure 3b), but had no effect on flower number (p = .825;Figure 3d).

| The relationships of individual size with reproductive allocation and total flower weight
We found that individual size had no effect on reproductive allocation (p = .486;Figure S2a).The was a significant relationship between individual size and total flower weight (p < .001; Figure S2b).

| The trade-off between flower size and number
We found that there was a marginally significantly negative correlation between flower size and number in G. lawrencei var.farreri (p = .051;Figure 4a).When controlling for variation in individual size, there was a significant trade-off between flower size and number (p < .001; Figure 4b).

| Results of the SEM
According to the parameter values, the SEM adequately fitted the data (Fisher's C = 10.551,p = .103,df = 6).The results of the SEM (Figure 5) showed that: (1) there was a significant negative corre-

| DISCUSS IONS
We explored the variation patterns in individual size, reproductive allocation, flower size, and flower number, as well as their Plant vegetative growth and reproduction promote and restrict each other, and their response to environmental changes depends on the investment balance of plants in vegetative growth and reproduction (Zhang, 2004).The accumulated biomass of G. lawrencei var.farreri decreased sharply with increasing elevation, due to harsh environments (e.g., low temperatures and variability and poor predictability of climate) at high-altitude areas (Körner, 2003;Zhang et al., 2020).This is consistent with most experimental and theoretical research results (Coomes & Allen, 2007;Kiełtyk, 2021;Körner, 2003;Sigdel et al., 2023).We found that the reproductive allocation of G. lawrencei var.farreri was relatively constant and did not increase or decrease with changes in elevation.This is inconsistent with most empirical and theoretical studies which suggested plant reproductive allocation increased with elevation (Fabbro & Körner, 2004;Rathee et al., 2021).A previous study showed a significant positive correlation between reproductive allocation and elevation for annual and biennial plants, while the correlation between the two was not significant in perennial plants (Zhang et al., 2020).
For perennial plants (e.g., G. lawrencei var.farreri), the trade-off between current and future reproduction may mask the trade-off between vegetative growth and reproductive growth (Zhang, 2004).reproductive allocation not showing an upward trend with elevation, or even showing a downward trend.Finally, this study found that there was a trade-off between flower size and number in G. lawrencei var.farreri, which may also replace the trade-off between vegetative growth and reproduction to some extent, in order to adapt to highaltitude environmental conditions (see later discussion).
We also found that the reproductive allocation of G. lawrencei var.
farreri did not increase or decrease with changes in individual size.This is inconsistent with previous empirical and theoretical studies (reviewed by Zhang &Jiang, 2002 andZhang, 2004).These previous studies suggested that the larger the individual, the smaller the resources invested in the reproductive part.The decrease in reproductive allocation with increasing individual size may be a direct result of the increase in reproductive costs which can be partially explained by an increase in the allocation of reproductive support structures with increasing individual size (Reekie, 1998).In our study, the relatively smaller individuals of G. lawrencei var.farreri tended to grow at higher elevations, thus facing greater potential risks and resulting in higher reproductive costs.This may potentially lead to results in this study that reproductive allocation was not related to individual size.
The flower size of entomophilous plants affects plant attractiveness to pollinators (Krizek & Anderson, 2013;Teixido et al., 2018), which in turn affects plant reproductive success (Hou et al., 2024;Wei et al., 2021).In alpine environments, as elevation increases, environmental variables such as temperature, growth season length, and resource availability decrease, and the diversity, abundance, and activity ability of pollinating insects also decrease accordingly (Körner, 2003).The shortage of pollinators is the main selection pressure for the evolution of reproductive strategies in alpine plants (Duan et al., 2007;Sun et al., 2018).In high-altitude environments, plants may potentially increase their investment in male organs or attraction structures (Fabbro & Körner, 2004;Rathee et al., 2021), thereby reducing the impact of adverse factors such as the scarcity of pollinators, which is beneficial for improving plant pollination success rate.This may be the potential reason why we found that the flower size of the G. lawrencei var.farreri increased with elevation increasing.Meanwhile, we also found that the flower number of the G. lawrencei var.farreri decreased with elevation increasing.Therefore, the changes in biotic (pollination limitation) and abiotic (temperature and resource availability decreasing) environments along the elevation gradient jointly derived the trade-off between flower size and number in G. lawrencei var.farreri (Figure 4).This trade-off is of great significance in plant evolution and adaptation (Caruso et al., 2012;Sargent et al., 2007).In this study, the trade-off between flower size and number may potentially improve the adaptability of plants to climate change along an elevation gradient.The empirical evidence for this trade-off is mixed (reviewed by Caruso et al., 2012 andSargent et al., 2007).We found that the differences in plant resource acquisition ability potentially caused by individual size may mask the true trade-off relationship (Figure 4).
An interesting finding in this study is that the flower size of G. lawrencei var.farreri depended on reproductive allocation rather than individual plant size, and in contrast, flower number depended on individual plant size rather than reproductive allocation (Figure 3).That is to say, when plant reproduction allocation increased (i.e., the relative amount of reproduction increasing), plants tended to invest in flower size but not in flower number; when the individual size of a plant increased (equivalent to an increase in the absolute amount of reproduction; Figure S2b), the plant tended to invest in flower number but not in flower size.It is generally believed that the size and number of flowers increase with individual size, and previous studies have confirmed this point (for flower size: Worley et al., 2000;Worley & Barrett, 2001; for flower number: Morgan, 1998;Sato & Yahara, 1999;Sun et al., 2018;Worley & Barrett, 2000).However, similar to several previous studies (Iwaizumi & Sakai, 2004;Sato & Yahara, 1999), we found that flower size did not increase with individual size.It might be explained by the trade-off between flower size and number, as well as the evidence that we found that individual size can increase the number of flowers.Through multiple statistical methods such as linear mixed-effect and structural equation model analyses, we found that flower size was controlled by the relative reproductive allocation rather than the absolute reproductive allocation, while flower number was controlled by the absolute reproductive allocation rather than the relative reproductive allocation.Our results provided a new perspective for this research question.Of course, more researches will be needed in the future to validate our results.

| CON CLUS IONS
We found that along an elevation of 3200-4000 m, G. lawrencei var.
size, reproductive allocation, flower size, and flower number.This study increases our understanding of the adaptation mechanisms of alpine plants to climate change in alpine environments.Plateau as the research object, mainly studying the relationship of flower size and number, as well as the effects of elevation, individual size, and plant reproductive allocation on flower size and number.We hypothesized that elevation could influence flower size and number directly and indirectly via individual size and reproductive allocation (Figure1).To our knowledge, there is no published research on the direct and indirect effect of elevation on flower size and number.Specifically, the objectives of our study were to determine: (1) the direct and indirect effect of elevation on flower size and number through its direct impacts on individual size and reproductive allocation, and (2) whether there was a trade-off between flower size and number (Figure1).
4000 m above sea level).We counted the number of flowers per individual, then divided each individual into flowers and the rest, dried them at 60°C for 48 h, and weighed them.Based on these data, we obtained the data of individual size (aboveground biomass), reproductive allocation (total flower weight/aboveground biomass), and flower size (total flower weight/number of flowers) and number (the number of flowers per individual).
tween elevation and individual size, reproductive allocation, flower size, and flower number.The relationships of individual size with reproductive allocation and total flower weight (i.e., the absolute amount of reproduction) were tested using linear mixed-effect models, respectively.Linear mixed-effect models were also used to examine the effects of individual size and reproductive allocation on flower size and number, F I G U R E 1 The hypothetical pathways of how elevation influences flower size and number through individual size and reproductive allocation.respectively.The plant population was used as a random factor in these models.We conducted a linear mixed-effect model to detect the relationship between flower size and number.The differences in plant resource acquisition ability potentially caused by different individual sizes may affect the trade-off between flower size and number.Therefore, we use residuals to control the changes in individual size.Specifically, the residuals of flower size on individual size and the residuals of flower number on individual size were used in bivariate regressions to examine the relationship between flower size and number, independent of variation in individual size, using the linear mixed-effect model.The plant population was used as a random factor in these models.We used the structural equation model (SEM) to explore the connections among elevation, individual size, reproductive allocation, flower size, and flower number.We hypothesized a path diagram where elevation regulates flower size and number directly or indirectly through individual size and reproductive allocation (Figure 1).The outputs of the SEM were produced based on the p-values of conditional independence tests combined into a single Fisher's C statistic.

3. 2 |
Relationships of individual size and reproductive allocation with flower size and numberWe found that individual size significantly positively affected flower number (p < .001;Figure3c), but had no effect on flower size (p = .321;Figure3a).Reproductive allocation significantly positively F I G U R E 2 Relationships between elevation and individual size (a), reproductive allocation (b), flower size (c), and flower number (d).
lation between flower size and number; (2) individual size directly and significantly affected flower number, while reproductive allocation directly and significantly affected flower size; (3) elevation negatively affected flower number directly or indirectly through individual size; (4) elevation directly affected flower size.These resultswere consistent with the results of linear models (Figures2, 3, and 4).In addition, the SEM explained 33%, 0%, 44%, and 80% variance in individual size, reproductive allocation, flower size, and flower number, respectively.

F
Relationships of individual size and reproductive allocation with flower size (a, b) and number (c, d).Residuals were used to control the changes in individual size.The residuals of flower size on individual size (i.e., residuals flower size) and the residuals of flower number on individual size (i.e., residuals flower number) were used in bivariate regressions to examine the relationship between flower size and number, independent of variation in individual size, using the linear mixed-effect model.relationships, of a typical alpine plant, G. lawrencei var.farreri, on an elevation gradient of 3200-4000 m in the northeast of the Qinghai-Tibet Plateau.We found that with elevation increasing, the individual size of G. lawrencei var.farreri became smaller, and the flowers became larger and fewer, while the reproductive allocation remained unchanged.There was a trade-off between flower size and number in G. lawrencei var.farreri, which reflects its adaptation to alpine biotic and abiotic environments.
In addition, as elevation increases, some ecological factors may improve (e.g., soil organic carbon often accumulating in large stocks in cold regions; García-Palacios et al., 2024), which may result in F I G U R E 4 The trade-off between flower size and number when not controlling (a) and controlling (b) for variation in individual size.Residuals were used to control the changes in individual size.The residuals of flower size on individual size (i.e., residuals flower size) and the residuals of flower number on individual size (i.e., residuals flower number) were used in bivariate regressions to examine the relationship between flower size and number, independent of variation in individual size, using the linear mixed-effect model.F I G U R E 5 Structural equation model (SEM) of the relationships among elevation, individual, reproductive allocation, flower size, and flower number.Solid yellow and green arrows indicate significant negative and positive effects (*p < .05;**p < .01;***p < .001),respectively.The numbers above arrows indicate path coefficients.The width of arrows indicates the strength of the causal influence.R 2 represents the proportion of variance explained for each dependent variable in the model (Fisher's C = 10.55,p = .103,df = 6).
farreri became smaller and its flowers became larger and fewer, while the reproductive allocation remained unchanged.The flower size of G. lawrencei var.farreri depended on reproductive allocation rather than individual plant size, while the flower number depended on individual plant size rather than reproductive allocation.There was a clear trade-off between flower size and number in G. lawrencei var.farreri, and individual size partially masked this trade-off.Elevation decreased flower number directly and indirectly via reducing individual size.And elevation and reproductive allocation both directly increased flower size.The variation patterns in individual size, reproductive allocation, flower size, and flower number of G. lawrencei var.farreri, as well as the trade-off between flower size and number, reflect its adaptation to alpine environments.This study increases our understanding of the adaptation mechanisms of alpine plants to climate change on the elevation gradient.AUTH O R CO NTR I B UTI O N S Mengyan Wang: Data curation (equal); investigation (equal); methodology (equal); software (equal); writing -original draft (equal).Zuoyi Wang: Data curation (equal); investigation (equal); methodology (equal); visualization (equal); writing -review and editing (equal).Yuan Yang: Data curation (equal); investigation (equal); visualization (equal);